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. 2012;7(11):e50491.
doi: 10.1371/journal.pone.0050491. Epub 2012 Nov 30.

Adult cardiac progenitor cell aggregates exhibit survival benefit both in vitro and in vivo

Michael Bauer  1 , Lifeng Kang, Yiling Qiu, Jinhui Wu, Michelle Peng, Howard H Chen, Gulden Camci-Unal, Ahmad F Bayomy, David E Sosnovik, Ali Khademhosseini, Ronglih Liao
Affiliations

Adult cardiac progenitor cell aggregates exhibit survival benefit both in vitro and in vivo

Michael Bauer et al. PLoS One. 2012.

Abstract

Background: A major hurdle in the use of exogenous stems cells for therapeutic regeneration of injured myocardium remains the poor survival of implanted cells. To date, the delivery of stem cells into myocardium has largely focused on implantation of cell suspensions.

Methodology and principal findings: We hypothesize that delivering progenitor cells in an aggregate form would serve to mimic the endogenous state with proper cell-cell contact, and may aid the survival of implanted cells. Microwell methodologies allow for the culture of homogenous 3D cell aggregates, thereby allowing cell-cell contact. In this study, we find that the culture of cardiac progenitor cells in a 3D cell aggregate augments cell survival and protects against cellular toxins and stressors, including hydrogen peroxide and anoxia/reoxygenation induced cell death. Moreover, using a murine model of cardiac ischemia-reperfusion injury, we find that delivery of cardiac progenitor cells in the form of 3D aggregates improved in vivo survival of implanted cells.

Conclusion: Collectively, our data support the notion that growth in 3D cellular systems and maintenance of cell-cell contact improves exogenous cell survival following delivery into myocardium. These approaches may serve as a strategy to improve cardiovascular cell-based therapies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cell patterning and aggregate formation inside microwells.
A) Cell patterning. Cells were localized inside the microwells. B) After cell seeding, the cells in the microwell array were cultured in a petri dish and aggregates formed within 24 h. C) Once the aggregate formation is complete inside the microwells, they can be stained. D) Aggregates can be imaged inside microwells. E) Aggregates can be easily released from the microwells by gentle flushing with media for other applications.
Figure 2
Figure 2. Characterization of aggregate formation and uniformity.
A) Time course of aggregate formation in the microwell at 0,1,2,3,4, and 12 h post seeding. B) Quantification of aggregates size over time (n = 2). C) Size of the aggregates can be controlled by seeding microwell arrays for CSP aggregation with different cell numbers in microwells. The cell suspension densities used for seeding were 4×106/mL, 8×106/mL, and 16×106/mL, resulting in aggregates with controllable sizes defined hereinafter as small aggregate (S), medium aggregate (M), and large aggregates (L), respectively. D) Subsets of a microwell array with CSP cell aggregates after 24 h showing high uniformity. E) CSP cells cultured in an ultra low adhesive (ULA) 96-well plate for 24 h. Cells form aggregates of various sizes. F) Frequency distribution of diameter of aggregates formed in microwells shows narrow distribution. The standard deviation (S.D.) of Gaussian distribution fitting is 10.9 µm. G) Frequency distribution of diameter of aggregates formed in ULA 96-well plates shows wide distribution. The S.D. of Gaussian distribution fitting is 77.7 µm. (All bars represent 100 µm).
Figure 3
Figure 3. Aggregate survival tests in vitro.
A) Subsets of microwell arrays with 2D monolayer of cell culture (2D) and aggregates of three sizes (S, M, and L). Hydrogen peroxide and anoxia/reoxygenation treatments were employed to induce cell death. EthD (red) and DAPI (blue) staining were performed for the determination of cell death. B) Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to 200 µM-hydrogen peroxide treatment using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes. C) Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to anoxia/reoxygenation using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes.
Figure 4
Figure 4. Aggregate integrity and survival in fluidic manipulations.
A) Aggregates formed in microwells can be easily flushed out from the microwell and centrifuged while remaining intact. B) Aggregate can be easily passed through a 30G needle without loosing integrity. C) A representative DAPI/EthD fluorescent image of aggregates before injection. D) A representative DAPI/EthD fluorescent image of aggregates after injection. E) Quantification of dead CSP cells in aggregates passing a 30G needle using EthD/DAPI fluorescent intensity ratio. (All bars represent 100 µm).
Figure 5
Figure 5. CSP cell survival in vivo following cardiac injury.
A) Protocol to measure the in vivo survival of CSP aggregates and suspensions. B) Representative serial bioluminescence images (BLI) of mice injected with CSP cell aggregates and CSP single cell suspensions. C) Percentage of CSP cell survival measured with BLI.
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